Phosphorothioate Nucleic Acids: Artificial Modification Envisaged by Nature

Internucleotide phosphate diester bonds in unmodified oligonucleotides are rapidly degraded by nucleolytic enzymes in cells or body fluids. This property excludes natural DNA and RNA molecules from potential medical applications and from many structural and mechanistic studies. DNA nucleotides and oligonucleotides in which one of the nonbridging phosphate oxygen atoms is replaced by a sulfur atom (PS-DNA) were among the first DNA analogs to be designed and synthesized. PS-DNA exhibits significantly higher nuclease resistance and also offers important opportunities for detailed studies of interactions with other biomolecules at the molecular level. However, the substitution creates a stereogenic center at the phosphorus atom, so that even short oligomers synthesized by a non-stereocontrolled method exist as mixtures of hundreds or even thousands of P-diastereomers, which usually cannot be separated chromatographically. Stereocontrolled synthesis methods have been developed to overcome this problem. P-stereodefined probes, including isotopomerically labeled species, have been used to elucidate the mode of action of numerous enzymes (nucleases, transferases, and kinases), ribozymes, and DNA-zymes, as well as to study the thermodynamic stability of nucleic acid complexes (duplexes, triplexes, and i-motif) and the mechanism of B-Z-type conformational changes. They are also useful tools for tuning the properties of siRNA duplexes. For many years, phosphorothioate modification was considered purely artificial, having been designed and implemented by chemists. However, in 2007, a phosphorothioate modification of DNA in bacteria was discovered and its functioning was intensively studied. In 2020, a report was published on the presence of a phosphorothioate modification in RNA isolated from prokaryotes and eukaryotes, but this claim has been criticized and seems premature, to say the least. This chapter covers two main areas related to PS-oligonucleotides: first, the synthetic routes to P-stereodefined oligonucleotides and selected examples of their application in structural, biochemical, and biological experiments; and second, the biosynthesis of oligonucleotides with phosphorothioate modification and their physiological functions.